The overall goals of this work are: 1) an understanding of the control of proliferative capacity in lymphoblastoid cells; and 2) elucidation of the mechanism controlling the production of "temperate" virus products in animal cells. We have found that the chemical mutagen/carcinogen methyl nitronitrosoguanidine will increase the frequency with which proliferative cultures can be obtained from normal peripheral blood lymphocytes. All such cultures carry the Epstein-Barr virus genome. Treatment of "immortalized" lymphoblastoid cells with methyl nitronitrosoguanidine results in an increased proportion of cells which produce viral antigens. We are studying the mechanism by which the mutagen produces its effects. Lymphoblastoid cells can carry as many as 50 copies of the EBV genome without producing viral products other than a nuclear antigen. Viral RNA may also be produced. It is clear that some factor exists in lymphoblastoid cells which inhibits viral transcription and/or translation. In order to isolate such factors we are attempting to develop an in vitro protein synthesizing system which will translate EB messenger RNA, or with the aid of RNA polymerase, EB-DNA. We are using an Ascites cell extract with rabbit reticulocyte initiation factors as the basis for our experiments. It will be necessary to recognize specific virus products and we are therefore continuing our studies on the EB-induced thymidine kinase. This enzyme is induced on viral infection, is stable in the presence of substrate at 60 degrees in contrast to cellular enzyme and has electrophoretic and kinetic properties which permit its recognition. We hope to transcribe specific virus products in vitro and then to study the in vivo regulatory system by addition of extracts of non-permissive cells to the functioning in vitro system.